ABSTRACT

In Medellin, Colombia, three Pseudomonas aeruginosa isolates with high-level carbapenem resistance (MIC ≥ 256 μg/ml) and an isolate of Citrobacter freundii with reduced susceptibility to imipenem produced the plasmid-mediated class A carbapenemase KPC-2. This is the first report of a KPC-type β-lactamase identified outside of the family Enterobacteriaceae.

The molecular class A carbapenemases of the KPC family (KPC-1 to -4) are a potent group of carbapenemases documented in numerous pathogens. While well identified in Klebsiella pneumoniae, these enzymes have been acknowledged throughout many members of the family Enterobacteriaceae, with reports in Klebsiella oxytoca (2, 27), Salmonella enterica (12), Escherichia coli (6, 14), Citrobacter freundii (6), Enterobacter spp. (3, 6, 7), and Serratia marcescens (6).

The KPC enzymes were once limited to sporadic occurrences in the eastern United States. They are now widespread in some facilities in New York and show an expanding geographic range (6). Sporadic identifications have also occurred on a global level, with reports of KPC-2 in four E. coli strains in Israel (14) and single isolates of K. pneumoniae from France (13) and China (24). We recently reported KPC-2 in two K. pneumoniae strains isolated from separate centers in Medellin, Colombia (22).

We now report isolates of P. aeruginosa and C. freundii producing KPC enzymes which were recovered from one of those same centers in Medellin, Colombia, where KPC-2 was identified in K. pneumoniae (22). Their identification in Pseudomonas represents the first report of KPC enzymes outside of the family Enterobacteriaceae.

(This work was presented in part at the 46th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, San Francisco, Calif., 2006, late breaker abstr. C2-421a.)

In January 2006, a tertiary care center in Medellin, Colombia, identified a C. freundii isolate resistant to imipenem by disk. The isolate was recovered from an aspirate of an intraabdominal abscess. The patient responded clinically to drainage. In April, in the same facility, three imipenem-resistant isolates of P. aeruginosa were recovered. Two of the isolates were from patients with ventilator-associated pneumonia. They received colistin and both subsequently died. The third P. aeruginosa isolate was from a surgical wound and appeared to reflect only colonization. All four patients had prolonged lengths of hospital stay, extensive antibiotic exposure, and were hospitalized concurrently between January and April 2006. No apparent association with the United States could be established in these patients.

The submitting hospital participates as part of a bacterial resistance surveillance network in Colombia. Therefore, the isolates were sent to the research facility Centro Internacional de Entrenamiento e Investigaciones Medicas (CIDEIM) for determination of the responsible resistance mechanism. Susceptibility testing by broth microdilution (4) confirmed the clinical lab findings. All three P. aeruginosa isolates showed unusually high-level β-lactam and carbapenem resistance (MICs ≥ 256 μg/ml) (Table 1). The isolates remained susceptible only to amikacin and colistin. In the C. freundii isolate, frank resistance occurred to ertapenem (MIC, 16 μg/ml), while reduced susceptibility was noted for imipenem and meropenem (MIC, 8 μg/ml). A two- to fourfold decrease in carbapenem MICs was seen with the addition of clavulanic acid for the Citrobacter isolate. No change was noted with clavulanic acid in P. aeruginosa, suggesting contributions to the carbapenem resistance by additional resistance mechanisms. Absence of OprD in all three isolates (data not shown) was confirmed by Western blot analysis (15) of outer membrane proteins (16), using an OprD-specific mouse monoclonal antibody (YD12; gift of Naomasa Gotoh, Kyoto Pharmaceutical University, Kyoto, Japan). Alterations in OprD expression affect imipenem and meropenem susceptibilities (8). Additionally, meropenem is affected by efflux overexpression; however, contributions by this mechanism were not examined.

Chromosomal DNA was fingerprinted by pulsed-field gel electrophoresis with XbaI digestion (Invitrogen, Carlsbad, CA) (11). The P. aeruginosa isolates revealed identical banding patterns, indicating clonal spread (data not shown). The high-level β-lactam resistance in Pseudomonas suggested a metallo-β-lactamase, a group which is commonly responsible for high-level resistance and has been found to occur in Colombian P. aeruginosa (5, 23). However, a PCR analysis for blaVIM and blaIMP (20) using genomic DNA was negative.

β-Lactamases in the cell extract from each isolate were evaluated by isoelectric focusing (10), with multiple and varied β-lactamases (pI range, 5.0 to 8.5) identified. One common pI ∼6.8 β-lactamase was identified in all isolates. A number of carbapenemases, including KPC-2 (22) and OXA-23 (J. N. Kattan, A. M. Guzman, A. Correa, S. Reyes, K. Lolans, N. Woodford, M. V. Villegas, J. P. Quinn and D. M. Livermore, Abstr. 46th Intersci. Conf. Antimicrob. Agents Chemother., abstr. C2-598, 2006) have been identified within the institution providing the isolates; both β-lactamases have isoelectric points in the pI 6.8 range. Thus, PCR analysis was performed with KPC (26) and OXA (25) primers. Surprisingly, the three Pseudomonas isolates as well as the C. freundii isolate were positive for blaKPC. Subsequent sequencing of the pCR-XL-TOPO (Invitrogen, Carlsbad, CA)-cloned amplicon from representative isolates (PA4036 and CF6010) confirmed blaKPC-2.

Attempts to transfer β-lactam resistance from both the P. aeruginosa and C. freundii representative isolates to E. coli J53 Azr by a mixed broth mating procedure (17) were unsuccessful. The genetic location for the blaKPC gene was investigated. Southern transfer of EcoR1- or BamH1-digested plasmids to a positively charged nylon membrane (Roche, Indianapolis, IN) (19) was performed. In every isolate, the blaKPC-2 probe (22) hybridized to a single band under high-stringency conditions (Fig. 1B). The BamHI digestion indicated that while similar blaKPC-containing plasmid fragments were identified among the P. aeruginosa isolates, the fragment differed in size from that in C. freundii. This difference in the plasmid profiles argues against conjugal transfer of a single plasmid between the species as facilitating the horizontal spread of this gene. A chromosomal location was investigated using the I-CeuI endonuclease technique (9). The KPC probe cohybridized with a single rRNA-hybridizing band from the representative P. aeruginosa, indicating a chromosomal location for this gene as well (Fig. 2, panel C). No similar hybridization of the KPC probe was identified for the C. freundii isolate. This is the first identification of a chromosomally encoded blaKPC. Further analysis of these plasmids and the genetic context associated with blaKPC is planned to further elucidate the mechanisms behind this gene dissemination.

Resistance rates for gram-negative bacilli tend to be much higher in Latin America than in the United States and Europe (18, 21). The carbapenems, retaining the broadest spectrum of activity of the β-lactams, are commonly considered the treatment of choice for infections resulting from multiresistant gram-negative organisms. P. aeruginosa possesses a larger array of resistance mechanisms than other organisms, which allows it to circumvent the activities of antimicrobial agents (1). The occurrence of the KPC enzyme in Klebsiella and Citrobacter and the emergence of an additional weapon in the pseudomonads' arsenal are serious concerns, especially as the future development of new antibiotics for gram-negatives looks bleak. The high-level carbapenem resistance seen in these Pseudomonas isolates, likely a result of both OprD loss and KPC production, may have facilitated the identification of the KPC enzyme. Accurate identification of carbapenem resistance mechanisms will help determine the epidemiology, risk factors, and appropriate therapeutic options for such strains.

ACKNOWLEDGMENTS

The conformation of the network of institutions that form the Colombian Nosocomial Resistance Study Group and funding for this study were made possible thanks in part to the support of the following: Bristol Myers Squibb, Colombia; Pfizer Inc., Groton, Conn.; Merck Research Laboratories, Whitehouse Station, N.J.; Baxter, Colombia; the Chicago Infectious Disease Research Institute, Chicago, Ill.